The invention is directed to tumor-susceptible non-human animals. The invention further pertains to the use of such animals in the development of anti-cancer agents and therapies.
Cancer is a set of diseases resulting from uncontrolled cell growth, which causes intractable pain and death for more than 300,000 people per year in the United States alone. Oncogenes and tumor suppressor genes are at opposite ends of a spectrum of gene actions that either promote or retard cancer cell growth. The development of cancer is believed to depend on the activation of oncogenes and the coincident inactivation of growth suppressor genes (Park, M., xe2x80x9cOncogenesxe2x80x9d in The Genetic Basis of Human Cancer (B. Vogelstein et al., eds.) pp. 205-228 (1998)). Oncogenes are mutated, dominant forms of cellular proto-oncogenes that stimulate cell proliferation, while tumor suppressor genes are recessive and normally inhibit cell proliferation (Cooper, 1995). The loss or inactivation of tumor suppressor genes is widely thought to be one of the contributors to unregulated cancer cell growth. While the discovery and identification of oncogenes has been relatively straightforward, identifying tumor suppressor genes has been much less so (Fearon, The Genetic Basis of Human Cancer (B. Vogelstein et al., eds.) pp. 229-236 (1998)).
Both oncogenes and tumor-suppressing genes have a basic distinguishing feature. The oncogenes identified thus far have arisen only in somatic cells and thus have been incapable of transmitting their effects to the germ line of the host animal. In contrast, mutations in tumor-suppressing genes can be identified in germ line cells and are thus transmissible to an animal""s progeny. About a dozen such tumor suppressor genes have been identified, with the hope that knowledge of their mechanism(s) might yield therapeutically relevant insights.
Tumor suppressor gene action depends on either mutation or deletion of both tumor suppressor alleles or on a reduction in the absolute level of expressed tumor suppressor protein. In their natural state, tumor suppressor genes act to suppress cell proliferation. Damage in such genes leads to a loss of this suppression, and thereby results in tumorigenesis. Knudson""s xe2x80x9ctwo-mutation hypothesisxe2x80x9d is a well studied statistical model for tumor suppressor gene action which is based on the epidemiological analysis of retinoblastoma. (Knudson, A. G., Proc. Nat. Acad. Sci. USA. 68:820-823 (1971)). According to this model, the host is heterozygous for the tumor suppressor gene, and cancer ensues when the single remaining functional allele also mutates to create a nullizygous state. An alternative model is the xe2x80x9chaplo-insufficient hypothesisxe2x80x9d in which the tumor cell produces abnormally low levels of wild type tumor suppressor gene product. Thus, in both of these models the deregulation of cell growth may be mediated by the inactivation of tumor-suppressing genes (Weinberg, R. A., Scientific Amer., September 1988, pp 44-51).
Tumor suppressor genes are principally known for control of cell proliferation by their action on the cell cycle. Well-studied examples include Rb (Weinberg, R. A., Cell, 81:323-330 (1996)), p53 (Greenblatt, M. S. et al., Cancer Res. 54:4855-4878, (1994); Williams, B. O. et al., Cold Spring Harbor Symp. Quant. Biol. 59:449, (1994)); Levine, A. J., Cell 88:323-331 (1997)), and p16 (Cairns, P., et al., Nat. Genetics 11:210-212, (1995)); Okamoto, A., et al., Cancer Research 55:1448-1451, (1995)). Another example of a tumor suppressor gene acting on the cell cycle is the p27KIP1 gene, also known simply as p27, which physiologically inhibits cyclin-dependent kinases, and thereby blocks cell proliferation (Fero, M. L., et al., Nature 396:177-180 (1998)).
In understanding how tumor suppressor genes impact the cell cycle, one must understand that cell cycle transitions are regulated by specific cyclin dependent kinases that consist of an activating cyclin subunit and a catalytic Cdk subunit (Polyak, K., et al., Cell 78:59-66 (1994)); Hartwell, L., Cell 71:543-546, (1992)); Nurse, P., Nature 344:503-508,(1990)). The functions of the respective cyclins and Cdk""s in mammalian cells correspond to the different phases of the cell cycle. For example, during the G1 phase, cyclin D-Cdk4/6 and cyclin E-Cdk2 are catalytically active and rate limiting for cell cycle progression. Growth factors induce the synthesis of D-type cyclins to initiate the G1 phase. The D-type cyclins then associate with Cdk4/Cdk6, and the active Cdk""s then hyperphosphorylate Rb to drive the cell past the restriction point (Buchkovich, K., et al., Cell 58:1097-1105 (1989)); see Weinberg, R. A., Cell 81:323-330 (1996)). Tumor suppressor genes have been found to affect the function of both of these types of subunits.
In addition to the cell cycle, tumor suppressor genes can also control cellular differentiation by acting as transcription factors and/or by modulating specific downstream DNA repair targets involved in maintaining genomic integrity. In this class, the tumor suppressor gene, inactivation of the tumor suppressor gene, p53, is the most common, resulting in a somatic mutation that causes malignancy (Nigro, J. M., et al., Nature 342:705-708 (1989); cf, review by Nguyen and Jameson, 1998). Of particular note, p53 is a frequent target for mutation in lung cancer (Takahashi, R., et al., Science 246:491-494 (1989)) and bladder cancers (Sidransky, D., et al., Science 252:706-709 (1991)). A germlne mutation for p53 is the basis for a familial cancer, the Li-Fraumeni syndrome (Srivastava, S., et al., Nature 348:747-749 (1990)). At the level of DNA repair, p53 works in the following manner: When DNA is damaged, a resulting signal causes stabilization of p53, which in turn causes transcriptional deregulation of p21, resulting in cell cycle arrest in the G1 phase (Hunter, T., Cell 75:839-841 (1993)).
Finally, tumor suppressor genes have also been implicated in controlling apoptotic cell death (Graeber, T. G., et al., Nature 379:88 (1996)). Again, p53 figures prominently in this process as well (Basu, A., et al., Mol. Hum. Reprod. 4:1099-1109 (1998)). The clear message from this brief summary is that the individual tumor suppressor genes cannot be viewed from a single perspective.
In order to study these tumor suppressor genes, model systems must be developed. Recent advances in recombinant DNA and genetic technologies have made it possible to discover and assess new tumor suppressor genes. One of the key model systems available is the transgenic animal. Such animals have been engineered to contain gene sequences that are not normally or naturally present in an unaltered animal. The techniques have also been used to produce animals which exhibit altered expression of naturally present gene sequences.
There remains a need for additional transgenic animals and methods of generating such animals, for the discovery of new tumor-suppressor genes.
The present invention provides a transgenic knockout mammal having somatic and germline cells comprising a chromosomally incorporated transgene. At least one allele of a genomic tumor suppressing annexin gene is disrupted by the transgene such that the expression of a tumor suppressing annexin gene is inhibited. This inhibition of the endogenous tumor suppressing annexin gene results in an increased susceptibility to formation of tumors as compared to a wild type mammal. The transgenic mammal may be heterozygous for this disruption. Preferably, the genomic tumor suppressing annexin gene is annexin VII. The preferred transgenic mammal is a transgenic rodent, and the more preferred transgenic mammal is a mouse.
Another embodiment of this method is the generation of transgenic embryonic stem cells. The method involves the steps of:
(a) constructing a transgene construct containing
(i) a recombination region having all or a portion of the endogenous tumor suppressing annexin gene and
(ii) a marker sequence which provides a detectable signal for identifying the presence of the transgene in a cell;
(b) transferring the transgene into embryonic stem cells of a mammal; and
(c) selecting embryonic stems cells having a correctly targeted homologous recombination between the transgene construct and the tumor suppressing annexin gene.
Another embodiment of the present invention comprises a method for generating a transgenic mammal having a functionally disrupted endogenous tumor suppressing annexin gene. The method involves the steps of:
(a) constructing a transgene construct containing
(i) a recombination region having all or a portion of the endogenous tumor suppressing annexin gene and
(ii) a marker sequence which provides a detectable signal for identifying the presence of the transgene in a cell;
(b) transferring the transgene into embryonic stem cells of a mammal;
(c) selecting embryonic stems cells having a correctly targeted homologous recombination between the transgene construct and the tumor suppressing annexin gene;
(d) transferring the cells of step (c) into a blastocyst and implanting the resulting chimeric blastocyst into a female mammal, and
(e) selecting those offspring harboring an endogenous tumor suppressing annexin gene allele comprising the correctly targeted recombination.
The preferred transgenic mammal for this method is a transgenic rodent, and the more preferred transgenic mammal is a transgenic mouse. The most preferred transgenic stem cell is a transgenic mouse stem cell. The most preferred tumor suppressing annexin gene is an annexin VII gene.
Another embodiment of the invention comprises a method for evaluating the carcinogenic potential of a test agent by contacting a transgenic mammal containing a disrupted tumor suppressing annexin gene with a test agent, and comparing the number of transformed cells in a sample of the treated transgenic mammal with the number of transformed cells in a sample from an untreated transgenic mammal. Alternatively, one can compare the number of transformed cells in a sample of the treated transgenic mammal with a control agent. The difference in the number of transformed cells in the treated transgenic mammal, compared to the number of transformed cells in the absence of treatment or in the presence of a control agent, indicates the carcinogenic potential of the test agent.
Another embodiment comprises a method of treating mammalian cancer cells lacking endogenous wild-type annexin protein, which comprises introducing a wild-type annexin tumor suppressor gene into the mammalian cancer cells, whereby the phenotype of abnormal proliferation of these mammalian cancer cells"" is suppressed by the expressed annexin protein. Preferably, the mammalian cancer cell lacks at least one allele of the wild-type annexin tumor suppressor gene. Preferably, the mammalian cancer cell is an osteosarcoma cell, lung carcinoma cell, lymphoma cell, leukemia cell, soft-tissue sarcoma cell, breast carcinoma cell, bladder carcinoma cell, or prostate carcinoma cell. More preferably, the mammalian cancer cell has a mutated annexin tumor suppressor gene.
Another embodiment comprises a method for treating a patient having a neoplasm characterized by abnormally proliferating cells in a mammal comprising administering an effective dose of a recombinant replication deficient virus comprised of a DNA segment that expresses a protein having the cell growth inhibition activity of the annexin tumor suppressor gene product. In one embodiment, the patient has a neoplasm comprised of cells that substantially lack a functional annexin tumor suppressor gene product. In another preferred embodiment, the neoplasm is comprised of cells that substantially lack a functional annexin VII gene product. Preferably, the replication-deficient virus is selected from the group consisting of a retrovirus, an adenovirus, a herpes simplex virus, a vaccinia virus, a papillomavirus, and an adeno-associated virus. Most preferably, the virus is a recombinant replication deficient adenovirus expression vector.
Another embodiment comprises a composition for therapy of a neoplastic disease characterized by the lack a functional annexin tumor suppressor gene product. The treatment comprises administering a therapeutically effective dose of a recombinant replication deficient adenovirus in a pharmaceutically deliverable form.
Another embodiment comprises a method of treating a disease characterized by abnormally proliferating cells in a mammal, by:
(a) administering an expression vector coding for an annexin protein to the mammal,
(b) inserting the expression vector into the abnormally proliferating cells, and
(c) expressing the tumor suppressor annexin gene in the abnormally proliferating cells in an amount effective to suppress proliferation of those cells.
Another embodiment is a DNA construct containing a recombination region having all or a portion of the endogenous tumor suppressing annexin gene and a marker sequence which provides a detectable signal for identifying the presence of the transgene in a cell. Preferably, the construct is KSBX.pPNT, as described below.
Another embodiment comprises a cell containing the DNA construct mentioned above. More preferably, the cell is a tumor cell, and most preferably, the cell is a mammalian cancer cell lacking endogenous wild-type annexin protein. In another preferable embodiment, the construct is KSBX.pPNT.
Another embodiment is an expression vector comprising an isolated polynucleotide sequence, which hybridizes to an annexin sequence under standard hybridization conditions and encodes a protein having the cell growth inhibition activity of an annexin protein. Preferably, the expression vector is selected from the group consisting of a retrovirus, an adenovirus, a herpes simplex virus, a vaccinia virus, a papillomavirus, and an adeno-associated virus. More preferably, the expression vector is a recombinant replication deficient adenovirus, and the polynucleotide sequence corresponds to the annexin VII gene.
Another embodiment comprises a cell transformed by the expression vector mentioned above.
Another embodiment comprises a method for identifying a polymorphism or a mutation in an exon of a human or animal tumor suppressor annexin VII gene. This method involves:
(a) incubating, under amplification conditions, a sample of genomic DNA comprising an exon of a human or animal tumor suppressor annexin gene with a primer pair comprising:
(i) a first primer which hybridizes to a promoter region or to an intron upstream of the exon, and
(ii) a second primer which hybridizes to the 3xe2x80x2-noncoding region or to an intron downstream of the exon, such that at least one primer of the primer pair hybridizes to an intron;
(b) producing an amplification product;
(c) determining the nucleotide sequence of the amplification product of the exon; and
(d) comparing the sequence of the exon obtained in step (b) to the sequence of a corresponding wild type exon.
A polymorphism or mutation is identified as a difference between these two sequences. Preferably, the exon is selected from the group consisting of exon 4, exon 5, exon 6, exon 7, and exon 8.
Another embodiment comprises a pharmaceutical preparation comprising an expression vector comprising an isolated polynucleotide sequence, which hybridizes to an annexin sequence under standard hybridization conditions and that encodes a protein having the cell growth inhibition activity of annexin VII, and a physiologically tolerable diluent.